Procedures for determining the presence or absence of specific organisms or viruses in a test sample commonly rely upon nucleic acid-based probe testing. To increase the sensitivity of these tests, an amplification step is often included to increase the number of potential nucleic acid target sequences present in the test sample. During amplification, polynucleotide chains containing the target sequence or its complement are synthesized in a template-dependent manner from ribonucleoside or deoxynucleoside triphosphates using nucleotidyltransferases known as polymerases. There are many amplification procedures in common use today, including the polymerase chain reaction (PCR), Q-beta replicase, self-sustained sequence replication (3SR), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), ligase chain reaction (LCR), strand displacement amplification (SDA) and loop-mediated isothermal amplification (LAMP), each of which is well known in the art. See, e.g., Mullis, “Process for Amplifying Nucleic Acid Sequences,” U.S. Pat. No. 4,683,202; Erlich et al., “Kits for Amplifying and Detecting Nucleic Acid Sequences,” U.S. Pat. No. 6,197,563; Walker et al., Nucleic Acids Res., 20:1691-1696 (1992); Fahy et al., “Self-sustained Sequence Replication (3SR): An Isothermal Transcription-Based Amplification System Alternative to PCR,” PCR Methods and Applications, 1:25-33 (1991); Kacian et al., “Nucleic Acid Sequence Amplification Methods,” U.S. Pat. No. 5,399,491; Davey et al., “Nucleic Acid Amplification Process,” U.S. Pat. No. 5,554,517; Birkenmeyer et al, “Amplification of Target Nucleic Acids Using Gap Filling Ligase Chain Reaction,” U.S. Pat. No. 5,427,930; Marshall et al., “Amplification of RNA Sequences Using the Ligase Chain Reaction,” U.S. Pat. No. 5,686,272; Walker, “Strand Displacement Amplification,” U.S. Pat. No. 5,712,124; Notomi et al., “Process for Synthesizing Nucleic Acid,” U.S. Pat. No. 6,410,278; Dattagupta et al., “Isothermal Strand Displacement Amplification,” U.S. Pat. No. 6,214,587; and Helen H. Lee et al., Nucleic Acid Amplification Technologies: Application to Disease Diagnosis (1997).
Because polymerase activity is readily lost at ambient temperature, it is common to manufacture amplification kits which include polymerases that have been freeze-dried in formulations containing other necessary co-factors and substrates for amplification. See, e.g., Shen et al., “Stabilized Enzyme Compositions for Nucleic Acid Amplification,” U.S. Pat. No. 5,834,254. Freeze-drying or lyophilization involves the removal of water from a frozen sample by sublimation under lower pressure. Sublimation is a process by which a solid is evaporated without passing through the liquid stage. Freeze-dried formulations containing polymerases are advantageous because they can be stored at ambient temperature and for prolonged periods of time without substantial losses of enzymatic activity.
Prior to use, dried polymerase formulations must be reconstituted with a reconstitution buffer, such as that disclosed by Shen et al., U.S. Pat. No. 5,834,254. Typically, the lyopholized product is provided in a vacuum-sealed glass bottle, and the buffer is separately provided in a plastic bottle or tube having a re-sealable cap. Reconstitution generally requires manually transferring the buffer from its container to the container holding the dried polymerase formulation, either by pipetting or pouring. The container holding the polymerase formulation is then swirled or otherwise agitated for a period of time sufficient to fully dissolve the dried material, after which time the reconstituted polymerase formulation is transferred back to the container which previously held the buffer. The container holding the reconstituted polymerase formulation is preferably a plastic container having a conically-shaped bottom to minimize waste when pipetting from the container. Plastic containers are preferred because they can be placed in sub-zero freezers for storage and are cheaper to manufacture than glass bottles. The reconstituted polymerase formulation may be used directly in an amplification procedure or sealed and stored for subsequent use.
The manual steps associated with commonly practiced polymerase reconstitution procedures raise two primary concerns. First, each of the manual steps involved in reconstituting dried polymerase formulations presents an opportunity for operator error and variability between reconstitutions, as the accuracy of reconstitutions depends upon precision pipetting or pouring by a practitioner. Second, open containers and manual transfer steps associated with such procedures provide an opportunity for practitioners to inadvertently contaminate reconstituted polymerase solutions with residual test material that may have been picked up from a laboratory workspace. This kind of contamination is especially undesirable since transferring even a minute amount of target-containing material from a workspace to a polymerase-containing solution could lead to the production of billions of target sequences in otherwise negative samples, thereby resulting in false-positives that would have tested negative in the absence of target amplification with the polymerases. Thus, it is an objective of the present invention to provide a manual method for reconstituting dried polymerase formulations in a manner which minimizes opportunities for operator error and contamination.